Despite extensive reliance on animal models, clinical translation in drug development remains limited, with over 90% of oncology candidates failing in human trials. To address this challenge, regulatory agencies and the pharmaceutical industry are increasingly adopting New Approach Methodologies (NAMs) that enhance human relevance while reducing animal use. Among these approaches, advanced in vitro liver models such as 3D hepatocyte spheroids offer improved physiological relevance compared to traditional systems, which are constrained by the rapid loss of metabolic activity and limited functional lifespan. In contrast, primary human hepatocytes cultured as 3D spheroids preserve cell–cell interactions, liver specific architecture, and enzymatic activity, enabling sustained viability and metabolic competence over multiple weeks. This extended functionality supports more accurate assessment of low-clearance compounds and emerging therapeutic modalities requiring prolonged exposure, positioning hepatocyte spheroids as a scalable and predictive platform for drug metabolism and toxicity studies.